Thanks to: Michael J. Freudiger

BiotechnologyRecombinant DNA and its

Applications

What will we be doing for the next three weeks?

1) You will be using restriction enzymes to cut plasmids.

2) You will be ligating the cut restriction fragments together to form recombinant plasmids.

3) You will be confirming that you created recombinant plasmids using gel electrophoresis.

4) You will be transforming E.coli bacteria with the recombinant plasmid.

5) You will be culturing the E.coli bacteria to determine if the correct transformation occurred.

6) You will be purifying the mFP protein produced by the transformed E.coli bacteria

Overview of what you already know

• At the beginning of the year you learned:

a)Micropipette Use

b)Loading an agarose gel

c)Reading the results of gel electrophoresis from the agarose gel

We will review these

Hold micropipette and epitubes at eye level

Micropipette UseMicropipette Use1. Twist dial to desired volumeTwist dial to desired volume2. Add disposable pipette tip. Add disposable pipette tip3. Press plunger to first stop . Press plunger to first stop 4. Insert pipette tip into solution to be . Insert pipette tip into solution to be transferredtransferred5. Slowly release plunger to retrieve liquid. Slowly release plunger to retrieve liquid6. Move pipette tip into desired tube. Move pipette tip into desired tube7. Press plunger past first stop to second stop. Press plunger past first stop to second stop to transfer liquid, watch liquid stick to wall to transfer liquid, watch liquid stick to wall of tube. Remove tip, then release plunger.of tube. Remove tip, then release plunger.8. Eject tip8. Eject tip

Reading a Pipette P-20

5.0 µL

For Lab 2,3,4: You will use P-20’s

Pipette Limits:

Never below 2.0 µL

Never above 20 µL

Reading a Pipette

P-20 P-200 P-1000

5.0 µL 50 µL 500 µL

Agarose Matrix:

Introduction to Gel Electrophoresis

Loading Gels:

Insert pipette tip:

•Under buffer level

•Above gel well

Micropipet tip should be ABOVE

the well NOT IN

IT!!!!

Micropipette tip punched

right through the

gel

See dye under the

wells

NICE!

Review of the how gel electrophoresis works

An electrical current AND a gel matrix are used to separate molecules.

Negatively charged molecules will migrate toward the positive side, and positively charged molecules will migrate toward the negative side.

The distance the molecules travel is based on several factors including:

1)Molecule size2)Molecule configuration3)Degree of charge on the molecule

Lab 1 Lab 2 Lab 3

Lab 4 Lab 5 Lab 6

Lab 1 Lab 2 Lab 3

Lab 4 Lab 5 Lab 6

Lab 1 Lab 2 Lab 3

Lab 4 Lab 5 Lab 6

What will be expected of you during this lab series

• Unlike other labs, part of your grade for this unit will be based on the result you achieve in lab #5 (growing a bacterial culture)

• You will also be working in groups of 3 (or 2), and be able to choose your partners.

• There will be specific assignments that will need to completed BEFORE and AFTER each lab

• There will be 2 quizzes between the labs, and a bigger quiz after all of the labs are completed

• In order to do well, you need to understand what will be happening BEFORE you actually perform the lab

Pre-Lab assignments

1) Create a flowchart of the lab you are about to do.

What is a flowchart?

A flowchart is a diagram of the actions that will happen in an activity.

Flowchart ExampleBelow is a flowchart for the series of labs we will be doing

Use restriction

enzymes to cut plasmids

Use restriction

enzymes to cut plasmids

Ligate the cut restriction

fragments together to form

recombinant plasmids

Ligate the cut restriction

fragments together to form

recombinant plasmids

Confirm you created

recombinant plasmids using gel

electrophoresis

Confirm you created

recombinant plasmids using gel

electrophoresis

Transform E.coli bacteria

with the recombinant

plasmid

Transform E.coli bacteria

with the recombinant

plasmid

Culture the E.coli bacteria to

determine if the correct

transformation occurred

Culture the E.coli bacteria to

determine if the correct

transformation occurred

Purify the mFP protein produced

by the transformed E.coli

bacteria

Purify the mFP protein produced

by the transformed E.coli

bacteria

Flowcharting

• Remember not to get bogged down in details

• Your flowchart should follow the steps you will be taking

• Use the written procedure for the details if you need them

• Flowchart will be stamped prior to the lab, and if revisions need to made, make them during the lab, right on your flowchart.

Pre-Lab assignments

2) Identify the following:

- What is the objective of the lab?

- What items do you start with?(Consumables & Hardware)

- What is produced at the end?(Be specific)

Pre-Lab assignments

3) Define important vocabulary terms

You will be given terms related to your lab, define these terms.

Important Vocabulary before we begin:

1)Aspirate: To draw up liquid

2)Dispense: To release liquid

3)Reagent: A chemical or compound

4)Microfuge Tube: The epitube or reagent tube

5)Plunger: The button on top of the micropipette

6)Aliquot: To measure out

Introduction

1) What are Plasmids?

2) How can we modify plasmids? Restriction Enzymes

3) Origins of restriction enzymes.

4) A close look at restriction enzymes.

5) Understanding plasmid diagrams.

What are Plasmids?

• Circular DNA that is used by bacteria to store their genetic information.

• Modifying plasmids to include extra genes allows for the production of new proteins.

In this Lecture…

How Can We Modify Plasmids?

1) Restriction Enzymes BamHI, HindIII, etc. Where do they come

from? How do they work? Different restriction

enzymes do different things.

2) DNA Ligase

Restriction Enzyme attached to DNA before cleavage

In this Lecture…

Origins of Restriction Enzymes

1) Bacteria produce restriction enzymes to protect against invading viral DNA/RNA.

Origins of Restriction Enzymes

2) The enzymes cut the invading DNA/RNA, rendering it harmless.

Restriction Enzyme in Action

1) DNA strand with EcoRI restriction site highlighted.

2) EcoRI restriction enzyme added (outline of separation about to occur).

3) Restriction fragments separate, with “sticky ends” at each edge.

Sticky Ends

Adding DNA Ligase

DNA ligase bonds sticky ends cut with the same restriction enzyme.

Sticky ends cut with different restriction enzymes will not bond together.

Why?

Because the base pair sequence of the two sticky ends will be different and not match up.

Sticky Ends

Plasmids Can Be Drawn to Show the Genes They Carry

In this diagram: • Blue and Orange are

drawn as genes.• Triangles are

indicating the known restriction sites for a restriction enzyme. (shapes can vary)

• Plasmid Maps are more complex.

Plasmid Name

Bp size

Plasmid Maps Indicate Restriction Sites and Genes

Make Recombinant DNA Using Restriction Enzymes

Application Exercise

DNA From Two Sources(Restriction Sites Labeled)

Circular DNA Linear DNA

Application of Restriction Enzymes

Adding DNA Ligase

Recombinant DNA Plasmid

• Many possible recombinant DNA plasmids can be produced, but this was the desired plasmid for the experiment.

Many Other Recombinant Possibilities

…and many more!

Running Digested DNA Through Gel Electrophoresis

Lab Experiment (Part 1)

Goals of this Hands-On Lab• Take plasmid DNA that has

been previously cut with restriction enzymes and compare that to a plasmid NOT cut with restriction enzymes, by running them through a gel.

• Look for different banding patterns and understand how to read them.

• Predict what kind of banding pattern a plasmid will make based on: 1. The restriction enzyme used.

2. The plasmid’s structural shape.

Gel Box Loading Techniques

• Look directly down the axis of the pipette.

• Loading dye makes the sample heavy, but it can still easily swish out of the well.

• Squirt down slowly.• Take the tip out of the buffer.• Then release the plunger.• If you don’t do that, you will

suck the sample back up.

Add DNA samples and ladder to the wells and “run to red!”

10 kb

8 kb

6 kb

5 kb

4 kb

3 kb

2 kb

1 kb

.5 kb

Sample fragments move toward positive end.

Analyzing Your Gel

Lab Experiment (Part 2)

What Makes Up the Banding Pattern in Restricted DNA?

• The restriction enzyme cleaves the DNA into fragments of various sizes.

• Each different size fragment will produce a different band in the gel.

• Remember that fragments separate into bands based on size.

Lancer Plasmid

6700 Bp

3300 Bp

2000 Bp

1400 Bp

What Makes Up the Banding Pattern After Adding DNA Ligase?

• Several combinations of plasmids will result from the reaction.

• The many forms will contribute to different bands.

(See following slides for chemical and structural forms)

Different Recombinant Forms

• Adding DNA Ligase does not always make the desired plasmid!

• Few if any could be what you wanted.

• Think about the large number of possible combinations.

Different Structural Forms

circle

“nicked-circle”

“multimer”

Different structural forms produce different bands.

Nicked Circle

SupercoiledLinear

10 K

b L

add

er

5 Kb

Multimer

NickedSuper Coiled

Linear Fragment

A- A+

Linear Fragment

10 K

b L

add

er

10 K

b L

add

er

Per 1 – Group 1

Per 1 – Group 2

Per 1 – Group 3

Per 1 – Group 4

Per 1 – Group 5

Per 1 – Group 6

Per 2 – Group 1

Per 2 – Group 2

Per 2 – Group 3

Per 2 – Group 4

Per 2 – Group 5

Per 2 – Group 6

Per 6 – Group 1

Per 6 – Group 2

Per 6 – Group 3

Per 6 – Group 4

Per 6 – Group 5

Per 6 – Group 6

Plasmid DNA Insertion

DNA plasmids can be inserted into bacteria using a variety of laboratory processes.

Transgenic Colony Allowed to Grow

How Do We Get the Desired Plasmid?• Restriction fragments will

ligate randomly, producing many plasmid forms.

• Bacterial insertion would be necessary, then colony growth, and further testing to isolate bacteria with the desired plasmid.

Transformation of bacterial cells through electroporation.

Bacteria are then moved to a growth plate, and grown on selective media to “weed out” cells that have not picked up the desired plasmid.

Recombinant plasmids

What Are Some Applications of Recombinant DNA Technology?

Bacteria, Yeasts, and Plants can all be modified to produce important pharmaceuticals, enriched foods, and industrial products.

Lab 7: Protein Purification

Honors Biology

Part 1: Obtaining a large concentration of cells with mFP

1. Get 1mL of cell culture2. Centrifuge for 5 min3. Identify location of red protein (mFP)4. Dump off supernatant5. Again add 1mL cells to the cell culture

tube6. Centrifuge for another 5 min, then dump

off the supernatant.

Freeing the Protein: Break open cells

Part 2: Breaking opened the cells

1. Obtain Elution buffer and lysis buffer from teacher

2. Add 150uL elution buffer to cells

3. Drag over tube rack to vortex (re-suspend cells in the solution)

4. Add 150uL lysis buffer to re-suspended cells

Freeing the Protein: Break open cells

• The protein is in living cells and mixed in with other proteins, over 1,000 proteins could be in one cell

• Pharmaceutical companies want one purified protein to sell as a medicine.

• Don’t want other proteins interfering with the medicine or body chemistry

Why purify a protein?

• We used an expression vector– The cells are making much more rfp than any

other protein

• Column chromatography:– We need to understand the amino acid make

up of the rfp• Hydrophobic and hydrophilic regions

With over 1,000 different proteins how can we isolate rfp?

• Hydrophobicity is often used to help separate molecules. – Hydrophobic: ‘fears water’ : oil, wax, fats– Hydrophilic: ‘loves water’ : salt, sugar

• Proteins have both hydrophobic and hydrophilic parts– Hydrophilic regions point outward– Hydrophobic regions point inward

Understanding purification Methods:

• mFP is highly hydrophobic.

• We use this property, to help purify the protein

• By putting mFP into a solution with a high salt concentration, we can distort the tertiary structure of the mFP

• Now, the hydrophobic regions point outward

Understanding purification Methods:

• Plastic column

• Buffer solution

• Hydrohpcic resin

• Stopcock

• Rfp

• Other proteins

How does chromatography work?

• By adding high salinity to the column the hydrophobic regions of the mFP stick to the hydrophobic resin in the column

• As you let the column drain, other protiens are washed out, rfp is stuck

Column Chromatography

• Equilibration buffer (2 M)– Column is stored in 20% ethanol, we are

washing out the ethanol to prepare column for our mFP

• Binding buffer (4 M salt solution)– Mix with mFP sample to make hydrophobic

regions turn outward: this way mFP will stick to resin

• Wash buffer (1.3 M)– Washes away “other” proteins but is still salty

enough to keep mFP holding on to resin• Elution buffer (10 mM)

– Makes hydrophobic regions of protein bend in, so mFP can’t hold onto resin

Lab 7 Column Buffers